Proper orientation of the mitotic spindle along an existing polarity axis is essential for cell differentiation, morphogenesis, and organogenesis. Spindle orientation in progenitor stem cells controls daughter cell fate and position during development and ensures homeostasis of adult tissues. Defects in oriented cell divisions are associated with neurodevelopmental disorders, polycystic kidneys, and cancer; thus, defining the molecular pathways that regulate spindle orientation has significant implications to human health. The long- term objective of our research program is to deduce the molecular mechanisms through which the actin and microtubule (MT) cytoskeletons control spindle orientation. The Wnt signaling effector, Disheveled (Dsh), regulates spindle orientation in several model systems, and we have recently identified Mushroom body Defect (Mud), together with the MT motor protein Dynein, as downstream Dsh components. Although the Mud/Dynein complex is required for spindle orientation, its function is not sufficient, suggesting involvement of unidentified alternative pathways. The specific objective of this proposal is to identify this alternative pathway and to establish a definitive molecular model for Dsh-mediated spindle orientation. We hypothesize that Dsh enhances cortical actin polymerization through a Rho/formin pathway, which provides a polarized spindle MT capture site and enhances localization of Mud/Dynein activators. This hypothesis is based on preliminary data demonstrating (a) Dsh-mediated asymmetric cortical actin staining and (b) loss of spindle orientation following inhibition of the actin regulator Rho. Although an involvement of the actin cytoskeleton in spindle orientation has long been speculated, a clearly defined role in animal cells remains controversial, as pleiotropic effects on cell polarity often occur upon inhibition of actin polymerization. My laboratory has recently developed an 'induced polarity' assay in Drosophila S2 cells that can circumvent nonspecific polarity defects while also allowing for high-throughput analysis of candidate pathway components. Furthermore, my laboratory has developed skill sets in biochemistry and Drosophila genetics that complement the discovery power of the S2 cell assay. Our combined approach offers an excellent experimental design for identifying the molecular basis for the actin cytoskeleton in Dsh-mediated spindle orientation. We will pursue our research objective through the following specific aims: (1) Characterize the molecular pathway for Dsh- mediated polarized actin polymerization required for spindle orientation, (2) Define the role of actin regulation in Drosophila sensory organ precursor (SOP) cell spindle orientation, and (3) Determine the molecular basis for DEP-CT domain synergy in Dsh-mediated spindle orientation. We believe this proposal will identify novel modes of spindle orientation that will further our understanding of human disorders associated with defective oriented cell division.